成纤维细胞生长因子20单克隆抗体的制备及鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-1525

摘要/Abstract

摘要：

构建表达重组人成纤维细胞生长因子20（fibroblast growth factor 20,FGF20）原核表达载体,并制备抗FGF20单克隆抗体,为开发新的药物奠定基础。构建pET24a-SUMO-hFGF20原核表达载体,转化到大肠杆菌BL21（DE3）中诱导表达。经镍NTA亲和层析柱纯化融合蛋白,SUMO酶酶切后,再次经镍NTA亲和层析柱纯化,SDS-PAGE法检测目的蛋白纯度。以重组FGF20蛋白免疫Balb/c小鼠,常规方法制备FGF20单克隆抗体,采用protein G柱分离纯化,SDS-PAGE法检测抗体纯度,ELISA法检测抗体效价和亚型,Western blot和SPR法测定抗体亲和力。成功构建pET24a-SUMO-hFGF20重组质粒。经分离纯化后获得FGF20蛋白相对分子质量为23 kD。得到1株ELISA检测强阳性杂交瘤细胞株,单克隆抗体纯化后,效价为1:25 600,亚型为IgG1,可以与商品化抗原结合,亲和力为1.07×10^-7mol/L。通过构建、表达和纯化重组蛋白,成功制备出抗FGF20蛋白高亲和力单克隆抗体。

关键词: 成纤维细胞生长因子20, 原核表达, 蛋白纯化, 单克隆抗体

Abstract:

Prokaryotic expression vector for expression recombinant fibroblast growth factor 20（FGF20）was constructed,and monoclonal antibody against FGF20 was prepared,which may lay foundation for developing novel medicines. The prokaryotic expression vector pET24a-SUMO-hFGF20 was constructed and transformed into Escherichia coli BL21（DE3）for induction and expression. The fusion protein was purified by Ni NTA affinity chromatography column,digested with SUMO enzyme,and purified again by Ni NTA affinity chromatography column. The purity of the protein was detected by SDS-PAGE. Balb/c mice were immunized with the recombinant FGF20 protein,and the monoclonal antibody was prepared by conventional way,and isolated and purified by protein G column. The purity of the antibody was detected by SDS-PAGE. The titer and subtype of the antibody were detected by ELISA. The affinity of the antibody was determined by Western blot and SPR. The recombinant plasmid pET24a-SUMO-hFGF20 was successfully constructed. The relative molecular weight of the isolated and purified FGF20 protein was 23 kD. A highly positive hybridoma cell line detected by ELISA was obtained. After purification,the titer of the monoclonal antibody was 1:25 600,and the subtype is IgG1. It bound to commercial antigen,and the affinity was 1.07×10 ^-7 M. High-affinity monoclonal antibody against the FGF20 protein was successfully prepared by construction,expression and purification of recombinant protein.

Key words: fibroblast growth factor 20（FGF20）, prokaryotic expression, protein purification, monoclonal antibody

唐禄, 董丽平, 尹茉莉, 刘磊, 董媛, 王会岩. 成纤维细胞生长因子20单克隆抗体的制备及鉴定[J]. 生物技术通报, 2021, 37(10): 179-185.

TANG Lu, DONG Li-ping, YIN Mo-li, LIU Lei, DONG Yuan, WANG Hui-yan. Preparation and Identification of a Novel FGF20 Monoclonal Antibody[J]. Biotechnology Bulletin, 2021, 37(10): 179-185.

图/表 8

图1 质粒双酶切图 M1:DL5 000 DNA marker;1:酶切前质粒;2:酶切后质粒;M2:DL2 000 DNA marker

Fig.1 Double enzyme digestion of plasmid M1:DL5 000 DNA marker. 1:Plasmid enzyme. 2:Plasmid unenzymed. M2:DL2 000 DNA marker

图2 工程菌可溶性分析电泳图 M:蛋白marker;1:诱导前;2:破碎沉淀;3:破碎上清

Fig.2 Electrophoregram analysis of soluble of engineere bacteria (SDS-PAGE) M:Protein marker. 1:Before induced. 2:Precipitation after crushing. 3:Supernatant after crushing

图3 FGF20纯化电泳图 M:蛋白marker ;1:SUMO酶切后;2:SUMO酶切前;3:FGF20

Fig.3 Electrophoregram analysis of purified FGF20 M:Protein marker. 1:Enzymed by SUMO. 2:Unenzymed by SUMO. 3:FGF20

图4 抗体纯化电泳图 M:蛋白marker;1:腹水;2:抗体

Fig.4 Electrophoregram analysis of purified monoclonal antibody M:Protein marker. 1:Ascites. 2:Purified antibody

图5 抗体效价分析检测

Fig.5 Antibody titer assay

图6 抗体亚型分析检测

Fig.6 Antibody subgroup assay

图7 Western Blot分析 1:纯化后抗原;2:商品化抗原

Fig.7 Western Blot assay 1:Purified antigen. 2:Commercial antigen

图8 SRP检测抗体与FGF20结合动力学特性

Fig.8 Kinetic assay on antibody binding to FGF20 antigen by SPR

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